CKI1, a Histidine Kinase Homolog Implicated in Cytokinin Signal Transduction
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Horgan R., Advanced Plant Physiology , Wilkins M. B. Pitman London 1984 53-75.
Skoog F., Miller C. O., Symp. Soc. Exp. Biol. 11 118 1957.
Arabidopsis (ecotype Wassilewskija) was transformed by the procedure of Akama K. , Shiraishi H. , Ohta S. , Nakamura K. and , Okada K. [Plant Cell Rep., 12, 7 (1992)], except that shoot-inducing medium was replaced by MS medium [ , Murashige >T. and , Skoog F., Physiol. Plant., 15, 473 (1962)] containing 1% sucrose, 1/100th volume of 5% 2-(N-morpholino)-ethanesulfonic acid (adjusted to pH 5.7 with KOH) myo-inositol (100 mg/liter), thiamine-HCl (20 mg/liter), pyridoxine-HCl (1 mg/liter), and nicotinic acid (1 mg/liter), solidified with 0.3% Phytagel (Sigma) as the basal medium for regeneration of transgenic organs and for mutant screening. Antibiotics and plant hormones were added as described (6) (Fig. 3).
The screenings for cytokinin-independent mutants were done as follows. Calli transformed with pPCVICEn4HPT were cultured on basal medium (5) supplemented with hygromycin (40 mg/liter) cefotaxime (150 mg/liter) and vancomycin (100 mg/liter) at 23°C under continuous light (5000 lux) for 3 weeks. Calli that proliferated rapidly turned green and produced shoots were selected and subcultured on the same medium. These lines were maintained by subculturing on basal medium (5).
Kakimoto T. data not shown.
DNA isolated from the cki1-1 line was digested with Spe I self-ligated and used to transform Escherichia coli (DH10B) by electroporation and the plasmid (pC1S1) was purified from the ampicillin-resistant E. coli cells as described elsewhere [;
Koncz C., et al., in Plant Molecular Biology Manual, , Gelvin S. B. and , Schilperoort R. A., Eds. (Kluwer Academic, Dordrecht, Netherlands, 1994), section B2].
A Spe I digest of pC1S1 was ligated to the Xba I fragment of pPCVICEn4HPT (4) which carries the T-DNA right border sequence the vegetative (oriV) the conjugational (oriT) DNA replication origins of plasmid RK2 and the T-DNA left border sequence to yield Ti plasmid pC1S1Ti.
RNA from the cki1-1 line was reverse-transcribed with the sequence-specific primer 5′-AGGCGTCCATTCCAGCTTGAATGG. The cDNA was amplified by polymerase chain reaction with a 5′ primer (5′-AGATCGCACCATTGTTGTTTGTAGC) and a 3′ primer (5′-CACACAAACCATACAGGCCAACCG) whose sequences were based on the genomic sequence. The amplified DNA was used to screen a cDNA library that had been constructed in Lambda ZAP II (Stratagene) in accordance with the manufacturer's instructions with polyadenylated [poly(A)+] RNA from shoot tissue of the cki1-1 line. Eighteen positive plaques were identified among 2 × 105 plaques and both ends of the five longest clones were sequenced. All of the sequences determined from the 5′ ends were found in pC1S1. Of the five clones cCKI1-16 was used for subsequent experiments. Both strands of cCKI1-16 were sequenced and the complete sequence of cCKI1-16 was present in pC1S1 with the exception of the 3′ noncoding region. cCKI1-16 contained 186 bp of 5′ noncoding sequence 3366 bp of coding sequence and 143 bp of 3′ noncoding sequence including poly(A). The DNA Data Bank of Japan accession number for the nucleotide sequence of the cDNA is D87545.
For construction of the Ti plasmid carrying CKI1 cDNA linked to the CaMV 35, S RNA promoter, 157 bp of the 5′ noncoding sequence of cCKI1-16 was removed by treatment with exonuclease III, and all of the 3′ noncoding sequence was removed by digestion with Spe I (the stop codon is at the Spe I site). The remaining sequence, together with the 34-bp vector sequences flanking the 5′ end of the cDNA, was blunt-end cloned into the Sma I site of pMON530 [ Rogers S. G. , Klee H. J. , Fraley R. B., Methods Enzymol. 153, 253 (1987)] next to the CaMV 35, S RNA promoter, and the plasmid that contained the insert in the sense orientation was selected and designated p35ScCKI1.
Five micrograms of poly(A)+ RNA was used for each lane.
The DNA region from the 5′ end of the Pst I site at nucleotide 2652 was used as a probe. Hybridization was done in 5× SSPE (750 mM NaCl 5 mM EDTA 50 mM sodium phosphate pH 7.4) that contained sheared salmon sperm DNA (200 μg/ml) and 0.2% SDS at 68°C; washing was done in 0.5× SSPE containing 0.2% SDS at 65°C.
Meyerowitz E. M., Methods in Arabidopsis Research , Koncz C., Chua N.-H., Schell J. World Scientific Singapore 1992 100-118.
Wilkinson J. Q., Lanahan M. B., Yen H.-C., Giovannoni J. J., Klee H. J., ibid. 270 1807 1995.
Schaller G. E., Bleeker A. B., ibid.: 1809.
Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
I thank D. Collings for correcting the English and for commenting on the manuscript H. Shibaoka for extensive support H. Hayashi for advice and Y. Shinozaki for critical reading of the manuscript. Special thanks are due to R. Walden for providing plasmid pPCVICEn4HPT for comments on the manuscript and for continuous encouragement. pMON530 was supplied by Monsanto. Supported in part by Grants-in-Aid for Scientific Research on Priority Areas (The Molecular Basis of Flexible Organ Plans in Plants number 06278103) and grants from the Nissan Foundation and the Sumitomo Foundation.